Process for the production of optically active beta-amino alcohols

ABSTRACT

A process for producing an optical active β-amino alcohol, the method comprising the step of allowing at least one microorganism selected from the group consisting of microorganisms belonging to the genus Morganella and others, to act on an enantiomeric mixture of an α-aminoketone or a salt thereof having the general formula (I):  
                 
 
     to produce an optical active β-amino alcohol with the desired optical activity having the general formula (II) described below in a high yield as well as in a highly selective manner:

TECHNICAL FIELD

[0001] This invention relates to a process for producing opticallyactive β-amino alcohols. More particularly, it relates to a process forproducing optically active β-amino alcohols which are of value as drugsor their intermediates.

BACKGROUND ART

[0002] Ephedrines have been used for purposes of perspiration,antipyresis and cough soothing from the olden times, and particularly,d-pseudoephedrine is known to possess anti-inflammatory action.Pharmacological action such as vasoconstriction, blood pressureelevation, or perspiration is known for 1-ephedrine and it is used intherapy as a sympathomimetic agent. 1-Ephedrine is also used in thetreatment of bronchial asthma. Specifically, processes for theproduction of optically active β-amino alcohols, including opticallyactive ephedrines, are useful in the manufacture of drugs and theirintermediates; thus, there is a need for efficient production processes.

[0003] In the conventional process for producing a β-amino alcohol withthe desired optical activity, there was used a process by which aracemic β-amino alcohol is obtained and then a specific optically activeform is produced by optical resolution or asymmetric synthesis amongothers.

[0004] However, since the racemic β-amino alcohol has two asymmetriccarbons within its molecule, complicated steps had to be followed toobtain the specific optically active form. For example, according toGer. (East) 13683 (Aug. 27, 1957), optically active phenylacetylcarbinolwas produced from benzaldehyde by fermentation utilizing yeast anderythro-1-2-methylamino-1-phenyl-1-propanol (i.e., 1-ephedrine) could beproduced by reductively condensing methylamine to the optically activephenylacetylcarbinol.

[0005] To obtain pseudoephedrine, the production is possible asdescribed in U.S. Pat. No. 4,237,304: an oxazoline is formed from1-ephedrine produced by the method described in Ger. (East) 13683 (Aug.27, 1957), using acetic anhydride, and then the oxazoline is hydrolyzedthrough inversion to the threo form (i.e., d-pseudoephedrine).

[0006] As stated above, to produce pseudoephedrine with the desiredoptical activity from 2-methylamino-1-phenyl-1-propanone, steps arenecessary such that ephedrine in the optical active erythro form is onceproduced and then it is inverted to the threo form. Hence, there ariseproblems that the number of steps grows and leads to complication andthat the yields lower.

[0007] Furthermore, in the production of the pseudoephedrine while asubstantial amount of diastereomers is produced as byproducts during thereduction of the starting ketone, the recovery of the diastereomers fortheir use as raw material is difficult, which is economicallydisadvantageous.

[0008] In addition, according to the method as described in thepublication of JP, 8-98697, A, it is possible to produce an opticallyactive 2-amino-1-phenylethanol derivative from a 2-amino-1-phenylethanolcompound having one asymmetric carbon atom within its molecule throughthe use of a specific microorganism. The present state of art is,however, that there has been no efficient process for producing β-aminoalcohol having two asymmetric carbon atoms.

DISCLOSURE OF THE INVENTION

[0009] This invention has been made in view of the above-indicatedcircumstances and it aims at producing a β-amino alcohol having thedesired optical activity from an enantiomeric mixture of anα-aminoketone compound or its salt in a high yield as well as in ahighly selective manner with a simple process while sufficientlypreventing the generation of diastereomeric byproducts.

[0010] The present inventors repeated studies diligently to solve theabove-stated problems; consequently, it was discovered that by utilizingspecific microorganisms only one enantiomer of the enantiomeric mixtureof an α-aminoketone compound or its salt could be reduced to produce theonly desired kind among the corresponding four kinds of β-amino alcoholsin a high yield as well as in a highly selective manner. This led to thecompletion of the present invention.

[0011] Specifically, the process for producing an optical active β-aminoalcohol according to this invention is a process for producing anoptical active β-amino alcohol characterized in that it allows at leastone microorganism selected from the group consisting of microorganismsbelonging to the genus Morganella, the genus Microbacterium, the genusSphingobacterium, the genus Nocardioides, the genus Mucor, the genusAbsidia, the genus Aspergillus, the genus Penicillium, the genusGrifola, the genus Eurotium, the genus Ganoderma, the genus Hypocrea,the genus Helicostylum, the genus Verticillium, the genus Fusarium, thegenus Tritirachium, the genus Mortierella, the genus Armillariella, thegenus Cylindrocarpon, the genus Klebsiella, the genus Aureobacterium,the genus Xanthomonas, the genus Pseudomonas, the genus Mycobacterium,the genus Sporobolomyces, the genus Sporidiobolus, the genusAmycolatopsis, the genus Coprinus, the genus Serratia, the genusRhodococcus and the genus Rhodotorula to act on an enantiomeric mixtureof an α-aminoketone or a salt thereof having the general formula (I):

[0012] wherein X may be the same or different and represents at leastone member selected from the group consisting of a halogen atom, loweralkyl, hydroxyl optionally protected with a protecting group, nitro andsulfonyl; n represents an integer of from 0 to 3; R¹ represents loweralkyl; R² and R³ may be the same or different and represent at least onemember selected from the group consisting of a hydrogen atom and loweralkyl; and “*” represents an asymmetric carbon,

[0013] to produce an optically active β-amino alcohol compound with thedesired optical activity having the general formula (II):

[0014] wherein X, n, R¹, R², R³ and “*” are as previously defined.

[0015] The microorganism according to this invention is preferably atleast one microorganism selected from the group consisting ofmicroorganisms belonging to Morganella morganii, Microbacteriumarborescens, Sphingobacterium multivorum, Nocardioides simplex, Mucorambiguus, Mucor javanicus, Mucor fragilis, Absidia lichtheimi,Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, Aspergilluscandidus, Aspergillus oryzae var. oryzae, Aspergillus foetidus var.acidus, Penicillium oxalicum, Grifola frondosa, Eurotium repens,Ganoderma lucidum, Hypocrea gelatinosa, Helicostylum nigricans,Verticillium fungicola var. fungicola, Fusarium roseum, Tritirachiumoryzae, Mortierella isabellina, Armillariella mellea, Cylindrocarponsclerotigenum, Klebsiella pneumoniae, Aureobacterium esteraromaticum,Xanthomonas sp., Pseudomonas putida, Mycobacterium smegmatis,Mycobacterium diernhoferi, Mycobacterium vaccae, Mycobacterium phlei,Mycobacterium fortuitum, Mycobacterium chlorophenolicum, Sporobolomycessalmonicolor, Sporobolomyces coralliformis, Sporidiobolus johnsonii,Amycolatopsis alba, Amycolatopsis azurea, Amycolatopsis coloradensis,Amycolatopsis orientalis lurida, Amycolatopsis orientalis orientalis,Coprinus rhizophorus, Serratia marcescens, Rhodococcus erythropolis,Rhodococcus rhodochrous and Rhodotorula aurantiaca.

[0016] In this invention the microorganism is preferably at least onemicroorganism selected from the group consisting of microorganismsbelonging to the genus Morganella, the genus Microbacterium, the genusSphingobacterium, the genus Nocardioides, the genus Mucor, the genusAbsidia, the genus Aspergillus, the genus Penicillium, the genusGrifola, the genus Eurotium, the genus Ganoderma, the genus Hypocrea,the genus Helicostylum, the genus Verticillium, the genus Fusarium, thegenus Tritirachium, the genus Mortierella, the genus Armillariella, thegenus Cylindrocarpon, the genus Klebsiella, the genus Aureobacterium,the genus Xanthomonas, the genus Pseudomonas, the genus Mycobacterium,the genus Sporobolomyces, the genus Sporidiobolus and the genusRhodococcus. More specifically, it is preferably a microorganismselected from the group consisting of microorganisms belonging toMorganella morganii, Microbacterium arborescens, Sphingobacteriummultivorum, Nocardioides simplex, Mucor ambiguus, Mucor javanicus, Mucorfragilis, Absidia lichtheimi, Aspergillus awamori, Aspergillus niger,Aspergillus oryzae, Aspergillus candidus, Aspergillus oryzae var.oryzae, Aspergillus foetidus var. acidus, Penicillium oxalicum, Grifolafrondosa, Eurotium repens, Ganoderma lucidum, Hypocrea gelatinosa,Helicostylum nigricans, Verticillium fungicola var. fungicola, Fusariumroseum, Tritirachium oryzae, Mortierella isabellina, Armillariellamellea, Cylindrocarpon sclerotigenum, Klebsiella pneumoniae,Aureobacterium esteraromaticum, Xanthomonas sp., Pseudomonas putida,Mycobacterium smegmatis, Mycobacterium diernhoferi, Mycobacteriumvaccae, Mycobacterium phlei, Mycobacterium fortuitum, Mycobacteriumchlorophenolicum, Sporobolomyces salmonicolor, Sporobolomycescoralliformis, Sporidiobolus johnsonii, Rhodococus erythropolis andRhodococcus rhodochrous. By utilizing such microorganisms, (1S,2S)-aminoalcohols tend to be obtained in simple processes as the optically activeβ-amino alcohols represented by the general formula (II) in high yieldsas well as in a highly selective manner.

[0017] Further, the microorganism is preferably at least onemicroorganism selected from the group consisting of microorganismsbelonging to the genus Amycolaptopsis, the genus Coprinus, the genusSerratia, the genus Rhodococcus and the genus Rhodotorula. Morespecifically, it is preferably a microorganism selected from the groupconsisting of microorganisms belonging to Amycolatopsis alba,Amycolatopsis azurea, Amycolatopsis coloradensis, Amycolatopsisorientalis lurida, Amycolatopsis orientalis orientalis, Coprinusrhizophorus, Serratia marcescens, Rhodococcus erythropolis, Rhodococcusrhodochrous and Rhodotorula aurantiaca. By utilizing suchmicroorganisms, (1R,2R)-amino alcohols tend to be obtained in simpleprocesses as the optically active β-amino alcohols represented by thegeneral formula (II) in high yields as well as in a highly selectivemanner.

[0018] Still further, in this invention the microorganism may becultured in a medium to which there has been added an activity inducerhaving the general formula (III):

[0019] wherein R⁴ represents lower alkyl; R⁵ and R⁶ may be the same ordifferent and each represents a hydrogen atom, lower alkyl or acyl; andY represents C═O or CH—OH. The mediation of such an activity inducerrenders the production of an optically active β-amino alcohol moreefficient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIGS. 1A through 1D are representations showing the structures ofthe optically active β-amino alcohols described below, including theirabsolute configurations.

[0021]FIG. 1A shows a β-amino alcohol with the (1S, 2S) configurationobtained according to this invention.

[0022]FIG. 1B shows (1S,2S)-(+)-pseudoephedrine obtained according tothe invention. FIG. 1C shows a β-amino alcohol with the (1R, 2R)configuration obtained according to the invention. FIG. 1D shows (1R,2R)-(−)-pseudoephedrine obtained according to this invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0023] The preferred embodiments of this invention will be described indetail hereafter.

[0024] The process for producing an optical active β-amino alcoholaccording to this invention is a process for producing an optical activeβ-amino alcohol characterized in that it allows at least onemicroorganism selected from the group consisting of microorganismsbelonging to the genus Morganella, the genus Microbacterium, the genusSphingobacterium, the genus Nocardioides, the genus Mucor, the genusAbsidia, the genus Aspergillus, the genus Penicillium, the genusGrifola, the genus Eurotium, the genus Ganoderma, the genus Hypocrea,the genus Helicostylum, the genus Verticillium, the genus Fusarium, thegenus Tritirachium, the genus Mortierella, the genus Armillariella, thegenus Cylindrocarpon, the genus Klebsiella, the genus Aureobacterium,the genus Xanthomonas, the genus Pseudomonas, the genus Mycobacterium,the genus Sporobolomyces, the genus Sporidiobolus, the genusAmycolatopsis, the genus Coprinus, the genus Serratia, the genusRhodococcus, and the genus Rhodotorula to act on an enantiomeric mixtureof an α-amino ketone compound or a salt thereof having the generalformula (I):

[0025] wherein X may be the same or different and represents at leastone member selected from the group consisting of a halogen atom, loweralkyl, hydroxyl optionally protected with a protecting group, nitro andsulfonyl; n represents an integer of from 0 to 3; R¹ represents loweralkyl; R² and R³ may be the same or different and represent at least onemember selected from the group consisting of a hydrogen atom and loweralkyl; and “*” represents an asymmetric carbon,

[0026] to produce an optically active β-amino alcohol compound with thedesired optical activity having the general formula (II):

[0027] wherein X, n, R¹, R², R³ and “*” are as previously defined.

[0028] The starting material used in the process for producing anoptically active β-amino alcohol according to this invention is anenantiomeric mixture of an α-aminoketone compound or a salt thereofhaving the general formula (I) wherein X may be the same or differentand represents at least one member selected from the group consisting ofa halogen atom, lower alkyl, hydroxyl optionally protected with aprotecting group, nitro and sulfonyl; n represents an integer of from 0to 3; R¹ represents lower alkyl; R² and R³ may be the same or differentand represent at least one member selected from the group consisting ofa hydrogen atom and lower alkyl; and “*” represents an asymmetriccarbon.

[0029] The substituent group X contained in the α-amino ketone will bedescribed in the following: the halogen atoms include a fluorine atom, achlorine atom, a bromine atom and an iodine atom.

[0030] The lower alkyl groups are preferably alkyls of from one to sixcarbons and include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,s-butyl, t-butyl, pentyl, isopentyl, hexyl, and the like. These mayadopt either of straight chain and branched structures and may have as asubstituent, a halogen atom such as fluorine or chlorine, hydroxyl,alkyl, amino, or alkoxy.

[0031] For the protecting group of the hydroxyl optionally protectedwith a protecting group, there are mentioned, among others, the one thatcan be removed upon treatment with water, the one that can be removed byhydrogenation, the one that can be removed by a Lewis acid catalyst orthiourea. The protecting groups include acyl optionally having asubstituent, silyl optionally having a substituent, alkoxyalkyl, loweralkyl optionally having a substituent, benzyl, p-methoxybenzyl,2,2,2-trichloroethoxycarbonyl, allyloxycarbonyl, trityl and the like.

[0032] The acyl groups include acetyl, chloroacetyl, dichloroacetyl,pivaloyl, benzoyl, p-nitrobenzoyl, and the like; they may also have asubstituent such as hydroxyl, alkyl, alkoxy, nitro, a halogen atom, orthe like. The silyl groups include trimethylsilyl, t-butyldimethylsilyl,triarylsilyl, and the like; they may also have a substituent such asalkyl, aryl, hydroxyl, alkoxy, nitro, a halogen atom, or the like. Thealkyl groups include methoxymethyl, 2-methoxyethoxymethyl and the like.The lower alkyl groups include alkyls of from one to six carbons: thereare mentioned methyl, ethyl, propyl, isopropyl, butyl, isobutyl,s-butyl, t-butyl, pentyl, isopentyl, hexyl, and the like. These mayadopt either of straight chain and branched structures and may have asubstituent such as a halogen atom (including fluorine and chlorine),hydroxyl, alkyl, amino, or alkoxy.

[0033] The X may be nitro or sulfonyl, and specifically, methylsulfonylis mentioned among others.

[0034] In addition, the number n of X is an integer of from 0 to 3,preferably 0.

[0035] R¹ in the general formula (I) represents lower alkyl. Such loweralkyls are preferably alkyls of from one to six carbons: there arementioned methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl,t-butyl, pentyl, isopentyl, hexyl, and the like. These may adopt eitherof straight chain and branched structures.

[0036] R² and R³ represent a hydrogen atom or lower alkyl. The loweralkyls include alkyls of from one to six carbons: there are mentionedmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl,pentyl, isopentyl, hexyl, and the like. These may adopt either ofstraight chain and branched structures.

[0037] The salts of the α-aminoketone compound include the salts ofinorganic acids such as hydrochloride, sulfate, nitrate, phosphate, andcarbonate and the salts of organic acids such as acetate and citrate.

[0038] The α-aminoketone can readily be synthesized by halogenating theα-carbon of the corresponding 1-phenyl ketone derivative (e.g.,bromination) and substituting the halogen such as bromo for amine (Ger.(East) 11, 332, Mar. 12, 1956).

[0039] The microorganism according to this invention is that which acton the enantiomeric mixture of the a α-minoketone having the generalformula (I) or a salt thereof. Such microorganism is one selected fromthe group consisting of microorganisms belonging to the genusMorganella, the genus Microbacterium, the genus Sphingobacterium, thegenus Nocardioides, the genus Mucor, the genus Absidia, the genusAspergillus, the genus Penicillium, the genus Grifola, the genusEurotium, the genus Ganoderma, the genus Hypocrea, the genusHelicostylum, the genus Verticillium, the genus Fusarium, the genusTritirachium, the genus Mortierella, the genus Armillariella, the genusCylindrocarpon, the genus Klebsiella, the genus Aureobacterium, thegenus Xanthomonas, the genus Pseudomonas, the genus Mycobacterium, thegenus Sporobolomyces, the genus Sporidiobolus, the genus Amycolatopsis,the genus Coprinus, the genus Serratia, the genus Rhodococcus and thegenus Rhodotorula. Specifically, the preferred ones include Morganellamorganii IFO 3848, Microbacterium arborescens IFO 3750, Sphingobacteriummultivorum IFO 14983, Nocardioides simplex IFO 12069, Mucor ambiguus IFO6742, Mucor javanicus IFO 4570, Mucor fragilis IFO 6449, Absidialichtheimi IFO 4009, Aspergillus awamori IFO 4033, Aspergillus niger IFO4416, Aspergillus oryzae IFO 4177, Aspergillus oryzae IAM 2630,Aspergillus candidus IFO 5468, Aspergillus oryzae var. oryzae IFO 6215,Aspergillus foetidus var. acidus IFO 4121, Penicillium oxalicum IFO5748, Grifola frondosa IFO 30522, Eurotium repens IFO 4884, Ganodermalucidum IFO 8346, Hypocrea gelatinosa IFO 9165, Helicostylum nigricansIFO 8091, Verticillium fungicola var. fungicola IFO 6624, Fusariumroseum IFO 7189, Tritirachium oryzae IFO 7544, Mortierella isabellinaIFO 8308, Armillariella mellea IFO 31616, Cylindrocarpon sclerotigenumIFO 31855, Klebsiella pneumoniae IFO 3319, Aureobacteriumesteraromaticum IFO 3751, Xanthomonas sp. IFO 3084, Pseudomonas putidaIFO 14796, Mycobacterium smegmatis IAM 12065, Mycobacterium diernhoferi,IFO 14797, Mycobacterium vaccae IFO 14118, Mycobacterium phlei IFO13160, Mycobacterium fortuitum IFO 13159, Mycobacterium chlorophenolicumIFO 15527, Sporobolomyces salmonicolor IFO 1038, Sporobolomycescoralliformis IFO 1032, Sporidiobolus johnsonii IFO 6903, Amycolatopsisalba IFO 15602, Amycolatopsis azurea IFO 14573, Amycolatopsiscoloradensis IFO 15804, Amycolatopsis orientalis lurida IFO 14500,Amycolatopsis orientalis orientalis IFO 12360, IFO 12362, IFO 12806,Coprinus rhizophorus IFO 30197, Serratia marcescens IFO 3736,Rhodococcus erythropolis IFO 12540, Rhodococcus erythropolis MAK-34,Rhodococcus rhodochrous IFO 15564, Rhodococcus rhodochrous IAM 12126,Rhodotorula aurantiaca IFO 0951, and the like.

[0040] Such microorganisms according to this invention permit theproduction of the corresponding optically active β-amino alcoholcompounds having the general formula (II), said compound possessing thedesired optical activity.

[0041] In the general formula (II), X, n, R¹, R², R³ and * are the sameas those in the general formula (I). Further, the β-amino alcoholshaving the desired optical activity include (1S,2S)-amino alcohol,(1S,2R)-amino alcohol, (1R,2S)-amino alcohol and (1R,2R)-amino alcohol.

[0042] In this invention the microorganism is preferably at least oneselected from the group consisting of microorganisms belonging to thegenus Morganella genus, the genus Microbacterium, the genusSphingobacterium, the genus Nocardioides, the genus Mucor, the genusAbsidia, the genus Aspergillus, the genus Penicillium, the genusGrifola, the genus Eurotium, the genus Ganoderma, the genus Hypocrea,the genus Helicostylum, the genus Verticillium, the genus Fusarium, thegenus Tritirachium, the genus Mortierella, the genus Armillariella, thegenus Cylindrocarpon, the genus Klebsiella, the genus Aureobacterium,the genus Xanthomonas, the genus Pseudomonas, the genus Mycobacterium,the genus Sporobolomyces, the genus Sporidiobolus and the genusRhodococcus. More specifically, preferred is a microorganism selectedfrom the group consisting of microorganisms belonging to Morganellamorganii, Microbacterium arborescens, Sphingobacterium multivorum,Nocardioides simplex, Mucor ambiguus, Mucor javanicus, Mucor fragilis,Absidia lichtheimi, Aspergillus awamori, Aspergillus niger, Aspergillusoryzae, Aspergillus candidus, Aspergillus oryzae var. oryzae,Aspergillus foetidus var. acidus, Penicillium oxalicum, Grifolafrondosa, Eurotium repens, Ganoderma lucidum, Hypocrea gelatinosa,Helicostylum nigricans, Verticillium fungicola var. fungicola, Fusariumroseum, Tritirachium oryzae, Mortierella isabellina, Armillariellamellea, Cylindrocarpon sclerotigenum, Klebsiella pneumoniae,Aureobacterium esteraromaticum, Xanthomonas sp., Pseudomonas putida,Mycobacterium smegmatis, Mycobacterium diernhoferi, Mycobacteriumvaccae, Mycobacterium phlei, Mycobacterium fortuitum, Mycobacteriumchlorophenolicum, Sporobolomyces salmonicolor, Sporobolomycescoralliformis, Sporidiobolus johnsonii, Rhodococcus erythropolis andRhodococcus rhodochrous. By utilizing such microorganisms, (1S,2S)-aminoalcohols tend to be obtained in simple processes as the optically activeβ-amino alcohols represented by the general formula (II) in high yieldsas well as in a highly selective manner.

[0043] Furthermore, in this invention the microorganism is preferably atleast one selected from the group consisting of microorganisms belongingto the genus Amycolatopsis, the genus Coprinus, the genus Serratia, thegenus Rhodococcus and the genus Rhodotorula. More specifically, morepreferred is a microorganism selected from the group consisting ofmicroorganisms belonging to Amycolatopsis alba, Amycolatopsis azurea,Amycolatopsis coloradensis, Amycolatopsis orientalis lurida,Amycolatopsis orientalis orientalis, Coprinus rhizophorus, Serratiamarcescens, Rhodococcus erythropolis, Rhodococcus rhodochrous andRhodotorula aurantiaca. By utilizing such microorganisms, (1R,2R)-aminoalcohols tend to be obtained in simple processes as the optically activeβ-amino alcohols represented by the general formula (II) in high yieldsas well as in a highly selective manner.

[0044] In addition, the microorganisms according to this inventioninclude (1S,2S)-amino alcohol producing bacteria that selectivelyproduce (1S,2S) forms among the optically active β-amino alcoholcompounds and (1R,2R)-amino alcohol producing bacteria that selectivelyproduce (1R,2R) forms among the optically active β-amino alcoholcompounds.

[0045] By allowing the action of the (1S,2S)-amino alcohol producingbacteria, there can be obtained, for example,d-threo-2-methylamino-1-phenylpropanol(d-pseudoephedrine),d-threo-2-dimethylamino-1-phenylpropanol(d-methylpseudoephedrine),(1S,2S)-α-(1-aminoethyl)-benzylalcohol(d-norpseudoephedrine),(1S,2S)-1-(p-hydroxyphenyl)-2-methylamino-1-propanol,(1S,2S)-α-(1-aminoethyl)-2,5-dimethoxy-benzylalcohol,(1S,2S)-1-(m-hydroxyphenyl)-2-amino-1-propanol,(1S,2S)-1-(p-hydroxyphenyl)-2-amino-1-propanol,(1S,2S)-1-phenyl-2-ethylamino-1-propanol,(1S,2S)-1-phenyl-2-amino-1-butanol and(1S,2S)-1-phenyl-2-methylamino-1-butanol. By allowing the action of the(1R,2R)-amino alcohol producing bacteria, there can be obtained, forexample, l-threo-2-methylamino-1-phenylpropanol(l-pseudoephedrine),1-threo-2-dimethylamino-1-phenylpropanol(l-methylpseudoephedrine),(1R,2R)-α-(1-aminoethyl)-benzylalcohol(l-norpseudoephedrine),(1R,2R)-1-(p-hydroxyphenyl)-2-methylamino-1-propanol,(1R,2R)-α-(1-aminoethyl)-2,5dimethoxy-benzylalcohol,(1R,2R)-1-(m-hydroxyphenyl)-2-amino-1-propanol,(1R,2R)-1-(p-hydroxyphenyl)-2-amino-1-propanol,(1R,2R)-1-phenyl-2-ethylamino-1-propanol,(1R,2R)-1-phenyl-2-amino-1-butanol and(1R,2R)-1-phenyl-2-methylamino-1-butanol.

[0046] Additionally, the obtained(1S,2S)-1-(m-hydroxyphenyl)-2-amino-1-propanol can be inverted toproduce (1R,2S)-1-(m-hydroxyphenyl)-2-amino-1-propanol (metaraminol).

[0047] Among the microorganisms according to this invention, those towhich IFO accession numbers have been designated are described in the“List of Cultures, 10th Edition (1966)” published by Institute forFermentation (IFO) (non-profit organization) and are available from theIFO. The microorganisms to which IAM accession numbers have beendesignated are described in the “Catalogue of Strains, 1993” publishedby Institute of Molecular and Cellular Biosciences, the Cell &Functional Polymer General Center, University of Tokyo and are availablefrom its preservation facilities. Further, Rhodococcus erythropolisMAK-34 is a novel microorganism isolated from the nature and has beendeposited with National Institute of Bioscience and Human-Technology,National Institute of Advanced Science and Technology, METI locating at1-3, Higashi 1-Chome, Tsukuba, Ibaraki, JAPAN (postal code: 305-8566) asFERM BP-7451 (the date of original deposit: Feb. 15, 2001).

[0048] For the microorganism used in this invention, there can be usedany of wild-type strains, mutant strains and recombinant strains derivedby the techniques of cell engineering such as cell fusion or by thetechniques of genetic engineering such as gene manipulations insofar asit is a microorganism capable of acting on the enantiomeric mixture of aα-aminoketone compound of the general formula (I) and producing thecorresponding optically active β-amino alcohol of the general formula(II).

[0049] There are no particular limitations to the different conditionsin the culturing of the microorganisms, and the methods that areordinarily used may be carried out, where bacteria, fungi, and yeast arecultured in suitable media, respectively. Normally, there may be usedliquid media containing carbon sources, nitrogen sources and othernutrients. Any sources may be used for the carbon source of the mediumas long as the microorganisms can utilize them. Specifically, there maybe used sugars such as glucose, fructose, sucrose, dextrin, starch, andsorbitol; alcohols such as methanol, ethanol, and glycerol; organicacids such as fumaric acid, citric acid, acetic acid, and propionic acidand their salts; hydrocarbons such as paraffin; and mixtures of theforegoing. Any sources may be used for the nitrogen source of the mediumas long as the microorganisms can utilize them. Specifically, there maybe used the ammonium salts of inorganic acids such as ammonium chloride,ammonium sulfate, and ammonium phosphate; the ammonium salts of organicacids such as ammonium fumarate and ammonium citrate; the salts ofnitric acid such as sodium nitrate and potassium nitrate;nitrogen-containing inorganic or organic compounds such as beef extract,yeast extract, malt extract, and peptone; and mixtures of the foregoing.Nutrition sources may also be added appropriately to the medium, whichare used in the normal culturing, including inorganic salts, the saltsof minute metals, and vitamins. There may also be added to the medium, asubstance for inducing the activity of a microorganism, a buffersubstance effective to maintain pH, or the like.

[0050] The substances for inducing the activity of a microorganisminclude an activity inducer having the general formula (III):

[0051] wherein R⁴ represents lower alkyl; R⁵ and R⁶ may be the same ordifferent and each represents a hydrogen atom, lower alkyl, or acyl; Yrepresents C═O or CH—OH.

[0052] The lower alkyl and acyl groups include the ones previouslydefined respectively. Specifically, the preferred activity inducersinclude 1-amino-2-propanol, 1-amino-2-hydroxybutane,1-acetylamino-2-propanol, 1-methylamino-2-propanol,1-amino-2-oxopropane, 2-amino-3-hydroxybutane, and the like. Whenasymmetric carbons are present in these compounds, the compounds may beeither of optically active forms and racemates, and may appropriately beselected. The addition of these activity inducers to medium induces theactivity of microorganisms and the subsequent generation of opticallyactive β-amino alcohols to progress with higher efficiency as comparedto the case with no such addition. The activity inducers may be usedindividually, or may be used as a mixture of plural inducers. Theaddition levels of such activity inducers are desirably 0.01 to 10 wt. %relative to medium.

[0053] Culturing of microorganisms can be carried out under theconditions suited for their growth. Specifically, it can be done at thepH of medium being 3-10, preferably pH 4-9 and at a temperature of 0-50°C., preferably 20-40° C. The culturing of microorganisms can be carriedout under aerobatic conditions or anaerobatic conditions. The culturingtime is preferably from 10 to 150 hours and should be appropriatelydetermined for the respective microorganisms.

[0054] The reaction method in the production of β-amino alcoholsaccording to this invention is not particularly limited insofar as it isa method by which the microorganism acts on the enantiomeric mixture ofthe α-amino ketone compound having the general formula (I) or a mixturethereof to produce the corresponding optically active β-amino alcoholcompound having the general formula (II). The reaction is allowed tostart by mixing bacterial cells washed with buffer or water to a aqueoussolution of the starting α-aminoketone.

[0055] The reaction conditions can be selected from the range withinwhich the generation of the optically active β-amino alcohol compoundhaving the general formula (II) is not impaired. The quantity ofbacterial cell is preferably {fraction (1/100)} to 1000 times, and morepreferably {fraction (1/10)} to 100 times that of racemic aminoketone.The concentration of the racemic aminoketone that is a substrate ispreferably from 0.01 to 20%, and more preferably from 0.1 to 10%.Further, the pH of reaction solution is preferably from 5 to 9, and morepreferably from 6 to 8; the reaction temperature is preferably from 10to 50° C., and more preferably from 20 to 40° C. Still further, thereaction time is preferably from 5 to 150 hours and it should beappropriately determined for the respective microorganisms.

[0056] In order for the reaction to progress more efficiently, sugars(e.g., glucose), organic acids (e.g., acetic acid), and energysubstances (e.g., glycerol) may be added. These may be respectively usedalone or may be used as a mixture thereof. The level of addition ispreferably {fraction (1/100)} to 10 times that of substrate. Coenzymesor the like may also be added. Coenzymes such as nicotinamide adeninedinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH),nicotinamide adenine dinucleotide phosphate (NADP), and reducednicotinamide adenine dinucleotide phosphate (NADPH) can be used alone oras a mixture of the foregoing. The level of addition is preferably from{fraction (1/1000)} to ⅕ times that of the racemic aminoketone. Inaddition to these coenzymes coenzyme-regenerating enzymes such asglucose dehydrogenase may also be added; and the level of addition ispreferably from {fraction (1/100)} to 10 times that of the racemicaminoketone. Further, sugars (e.g., glucose), organic acids (e.g.,acetic acid), and energy substances (e.g., glycerol), coenzymes,coenzyme-regenerating enzymes, and substrates for thecoenzyme-regenerating enzymes may respectively be combined for use.These substances are naturally accumulated in bacterial cells, but wheretheir addition as required can increase the reaction rate and yield,they may be appropriately selected.

[0057] Furthermore, when a certain salt is added such that the reactionsolution may be as described above and the reaction solution is allowedto react under that condition, the racemization of the unreactedα-aminoketone isomer can be accelerated and its conversion to theenantiomer that serves as the substrate for microorganism can beprogressed more efficiently. This tends to produce the objective aminoalcohol in a high yield of 50% or more from the starting material.

[0058] The salts for accelerating the racemization of the unreactedα-aminoketones may be the salts of weak acids such as acetate, tartrate,benzoate, citrate, malonate, phosphate, carbonate, p-nitrophenolate,sulfite, and borate. Preferably, there are used phosphates (e.g., sodiumdihydrogenphosphate, potassium dihydrogenphosphate, and ammoniumdihydrogenphosphate), carbonates (e.g., sodium carbonate, sodiumhydrogencarbonate, potassium carbonate, and ammonium carbonate),citrates (e.g., sodium citrate, potassium citrate, and ammoniumcitrate), for example. These mixtures may also be used, and desirably,buffers with pH 6.0-8.0 are added to give final concentrations of from0.01 to 1 M. For example, in the case of a phosphate, sodiumdihydrogenphosphate and sodium monohydrogenphosphate are desirably mixedat a ratio of from 9:1 to 5:95.

[0059] The optically active α-amino alcohols produced by reaction may bepurified by conventional separation/purification means. For example,directly from the reaction solution or after the bacterial cells areseparated, optically active β-amino alcohols can be obtained by beingsubjected to normal purification methods such as membrane separation orextraction with an organic solvent (e.g., toluene and chloroform),column chromatography, concentration at reduced pressure, distillation,recrystallization, and crystallization.

[0060] The optical activity of the optically active β-amino alcohol thusproduced can be determined by high performance liquid chromatography(HPLC).

EXAMPLES

[0061] This invention will be described concretely by way of examples;however, the scope of the invention is not to be limited by theseexamples.

Preparation Example 1

[0062] Preparation of dl-2-methylamino-1-phenyl-1-propanone

[0063] Bromine (51.6 ml) was added dropwise to a mixture of1-phenyl-1-propanone (134 g), sodium carbonate (42 g) and water (200ml), and reaction was allowed to take place at 70° C. for 3 hours togive a reaction mixture. To the reaction mixture was added 40% aqueousmonomethylamine solution (350 ml). After allowing to react at 40° C. for1 hour, the reaction product was extracted into chloroform (1 l ). Thereaction product in the chloroform layer was then extracted with dilutehydrochloric acid (100 ml), and activated carbon (3 g) was added to theaqueous layer and filtrated. The filtrate was concentrated to givedl-2-methylamino-1-phenyl-1-propanone hydrochloride (89 g).

Example 1

[0064] Production of d-(1S,2S)-pseudoephedrine

[0065]Microbacterium arborescens IFO 3750 was inoculated to a medium (5ml) containing 1% glucose, 0.5% peptone, and 0.3% yeast extract, andshake-culturing was carried out at 30° C. for 48 hours. After thecultured solution was centrifuged to give bacterial cells, the cellswere placed into a test tube. To this was added 0.1 M sodium phosphatebuffer (pH 7.0, 1 ml) and suspended. To this was addeddl-2-methylamino-1-phenyl-1-propanone hydrochloride (1 mg) and reactionwas allowed to take place under shaking at 30° C. for 24 hours. Afterthe reaction, the reaction solution was centrifuged to remove thebacterial cells and the supernatant was subjected to HPLC to giveoptically active pseudoephedrine: μ Bondapakphenyl manufactured byWaters Inc.; diameter of 4 mm; length of 300 mm; eluent-0.05 M sodiumphosphate buffer (containing 7% acetonitrile); pH 5.0; flow rate of 0.8ml/min; and detection light wavelength at 220 nm.

[0066] The absolute configuration and optical purity were determinedwith HPLC (Column Sumichiral AGP manufactured by Sumika ChemicalAnalysis Service; diameter of 4 mm; length of 150 mm; 0.03 M sodiumphosphate buffer; pH 7.0; flow rate of 0.5 ml/min; and detection lightwavelength at 220 nm). Consequently, only d-pseudoephedrine was obtainedselectively, as shown in Table 1.

[0067] The produced amounts will be all shown in terms of the amounts ofconverted hydrochloride hereafter.

Examples 2 to 12

[0068] Production of d-(1S,2S)-pseudoephedrine

[0069] Except that the microorganisms shown in Table 1 were used inplace of Microbacterium arborescens IFO 3750, optically activepseudoephedrine was obtained similarly to Example 1. Consequently, onlyd-pseudoephedrine was obtained selectively, as shown in Table 1. TABLE 1Optical activity (%) Produced Example Microorganism d-pseudo l-pseudoamount No. genus IFO d-ephedrine l-ephedrine ephedrine ephedrine (mg/mL)Example 1 Microbacterium 3750 0 0 100 0 0.16 arborescens Example 2Klebsiella 3319 0 0 100 0 0.3 pneumoniae Example 3 Aureobacterium 3751 00 100 0 0.14 esteraromaticum Example 4 Xanthomonas sp. 3084 0 0 100 00.049 Example 5 Pseudomonas 14796 0 0 100 0 0.1 putida Example 6Mycobacterium IAM 0 0 100 0 0.24 smegmatis 12065 Example 7 Mycobacterum14797 0 0 100 0 0.25 diernhoferi Example 8 Mycobacterum 14118 0 0 100 00.28 vaccae Example 9 Mortierella 8308 0 0 100 0 0.15 isabellina Example10 Cyllindrocarpon 31855 0 0 100 0 0.09 sclerotigenum Example 11Sporidiobolus 6903 0 0 100 0 0.07 johnsonii Example 12 RhodococcusMAK-34 0 0 100 0 0.3 erythropolis

Examples 13 to 37

[0070] Production of d-(1S,2S)-pseudoephedrine

[0071] Except that the microorganisms shown in Table 2 were used inplace of Microbacterium arborescens IFO 3750, optically activepseudoephedrine was obtained similarly to Example 1. The producedamounts and optical purities of pseudoephedrine are shown In Table 2.TABLE 2 Microorganism produced amount optical purity (%) Example No.genus IFO (mg/ml) d-pseudoephedrine Example 13 Nocardioides simplex12069 0.35 99 Example 14 Mycobacterium phlei 13160 0.27 95.6 Example 15Mucor ambiguus 6742 0.07 93 Example 16 Mucor javanicus 4570 0.04 95Example 17 Mucor fragilis 6449 0.17 90 Example 18 Absidia lichtheimi4009 0.04 93 Example 19 Aspergillus awamori 4033 0.18 93 Example 20Aspergillus niger 4416 0.11 90 Example 21 Aspergillus oryzae 4177 0.1891 Example 22 Aspergillus candidus 5468 0.07 94 Example 23 Aspergillusoryzae IAM2630 0.08 92 Example 24 Aspergillus oryzae 6215 0.05 95 var.oryzae Example 25 Penicillium oxalicum 5748 0.06 94 Example 26 Grifolafrondosa 30522 0.08 92 Example 27 Eurotium repens 4884 0.08 92 Example28 Ganoderma lucidum 8346 0.05 92.2 Example 29 Hypocrea gelatinosa 91650.27 92.2 Example 30 Helicostylum 8091 0.27 93.2 nigricans Example 31Aspergillus foetidus 4121 0.43 91.9 var. acidus Example 32 Verticilliumfungicola 6624 0.10 92.7 var. fungicola Example 33 Fusarium roseum 71890.40 89.6 Example 34 Tritirachium oryzae 7544 0.34 92 Example 35Armillariella mellea 31616 0.28 91 Example 36 Sporobolomyces 1038 0.1495 salmonicolor Example 37 Sporobolomyces 1032 0.2 95 coralliformis

Example 38

[0072] Production of d-(1S,2S)-pseudoephedrine

[0073]Morganella morganii IFO 3848 was inoculated to a medium containing1% glucose, 0.5% peptone, and 0.3% yeast extract, and shake-culturingwas aerobically carried out at 30° C. for 48 hours. After this culturedsolution (5 ml) was centrifuged to give bacterial cells, the cells weredried in the air and the resulting dried bacterial cells were suspendedin 1 ml of 0.05 M Tris hydrochloric acid buffer (pH 7.5). To theaforementioned dried bacterial cell suspension were added glucose (50mg), glucose dehydrogenase (0.2 mg), NADP (0.6 mg), NAD (0.6 mg), anddl-2-methylamino-1-phenyl-1-propanone hydrochloride (10 mg) andreciprocation-shaking was carried out at 28° C. and at 300 rpm. Afterallowing to react for 48 hours, the reaction solution was measured forthe produced amount and the optical activity of pseudoephedrine by HPLCsimilarly to Example 1. Consequently, only d-pseudoephedrine wasobtained selectively, as shown in Table 3. TABLE 3 Optical purity (%)Produced Microorganism d-pseudo- l-pseudo- amount Example No. Genus IFOd-ephedrine l-ephedrine ephedrine ephedrine (mg/ml) Example 38Morganella morganii 3848 0 0 100 0 0.79

Example 39

[0074] Production of d-(1S,2S)-pseudoephedrine hydrochloride

[0075]Mycobacterium smegmatis IAM-12065 was inoculated to a mediumcontaining 1% glucose, 0.5% peptone, and 0.3% yeast extract, andshake-culturing was aerobically carried out at 30° C. for 48 hours.After the cultured solution (1 l) was centrifuged to give bacterialcells, the cells was suspended in 50 ml of water, and after addition ofdl-2-methylamino-1-phenyl-1-propanone hydrochloride (0.5 g),reciprocation-shaking was carried out at 30° C. and at 150 rpm. Onehundred hours after the start of shaking, 7.0 g/l of d-pseudoephedrinewas produced in the reaction solution. After the reaction solution wascentrifuged to remove the bacterial cells, the pH was adjusted to 12 orgreater by addition of sodium hydroxide. Methylene chloride (100 ml) wasadded to this reaction solution and the reaction product was extracted.The solvent was removed, hydrochloric acid was added, and thenconcentration to dryness yielded a hydrochloride salt. The hydrochloridesalt was dissolved by addition of ethanol and further addition of ethercrystallized the reaction product. Consequently, d-pseudoephedrinehydrochloride was obtained. The resulting d-pseudoephedrine crystals(0.32 g) were analyzed on HPLC (Column Sumichiral AGP manufactured bySumika Chemical Analysis Service; diameter of 4 mm; length of 150 mm;0.03 M sodium phosphate buffer; pH 7.0; flow rate of 0.5 ml/min;detection light wavelength at UV 220 nm) and the optical activity wasfound to be 100%.

Example 40

[0076] Production of d-(1S,2S)-methylpseudoephedrine

[0077]Mycobacterium smegmatis IAM-12065 was inoculated to a mediumcontaining 1% glucose, 0.5% peptone, and 0.3% yeast extract, andshake-culturing was aerobically carried out at 30° C. for 48 hours. Thecultured solution (1 l) was filtrated to give bacterial cells, theresulting cells were washed with water, and water was added to form 50ml of suspension. To the suspension was added 100 mg of2-dimethylamino-1-phenyl-1-propanone hydrochloride (2 g/l), andreciprocation-shaking was carried out at 30° C. and at 150 rpm for 48hours. When the reaction solution was analyzed on HPLC (ColumnSumichiral AGP manufactured by Sumika Chemical Analytical Center;diameter of 4 mm; length of 150 mm; 0.03 M sodium phosphate buffer; pH7.0; flow rate of 0.5 ml/min; detection light wavelength at UV 220 nm),it was found that d-(1S,2S)-methylpseudoephedrine was produced at 0.23g/l and its optical activity was 77%.

Example 41

[0078] Production of l-(1R,2R)-pseudoephedrine

[0079]Amycolatopsis alba IFO 15602 was inoculated to a medium containing1% glucose, 0.5% peptone and 0.3% yeast extract, and shake-culturing wasaerobically carried out at 30° C. for 48 hours. The cultured solution (5ml) was centrifuged to give bacterial cells. After the cells weresuspended in 1 ml of 0.1 M sodium phosphate (pH 7.0) anddl-2-methylamino-1-phenyl-1-propanone hydrochloride (1 mg) was addedthereto, reaction was allowed to take place by carrying outreciprocation-shaking at 30° C. and at 150 rpm for 48 hours. When thereaction solution was analyzed on HPLC (Column Sumichiral AGPmanufactured by Sumitomo Chemical Analytical Center Co. Ltd.; diameterof 4 mm; length of 150 mm; 0.03 M sodium phosphate buffer; pH 7.0; flowrate of 0.5 ml/min; detection light wavelength at UV 220 nm), it wasfound that 1-pseudoephedrine was produced selectively. The result isshown in Table 4.

Examples 42 to 46

[0080] Production of l-(1R,2R)-pseudoephedrine

[0081] Except that the microorganisms shown in Table 4 were used inplace of Amycolatopsis alba IFO 15602, optically active pseudoephedrinewas obtained similarly to Example 41. Consequently, onlyl-pseudoephedrine was obtained selectively, as shown in Table 4. TABLE 4Optical activity (%) Produced Microorganism d- l- d-pseudo l-pseudoamount Example No. genus IFO ephedrine ephedrine ephedrine ephedrine(mg/ml) Example 41 Amycolatopsis alba 15602 0 0 0 100 0.33 Example 42Amycolatopsis 14573 0 0 0 100 0.064 azurea Example 43 Amycolatopsis15804 0 0 0 100 0.5 coloradensis Example 44 Amycolatopsis 14500 0 0 0100 0.18 orientalis lurida Example 45 Amycolatopsis 12360 0 0 0 100 0.5orientalis orientalis Example 46 Serratia marcescens 3736 0 0 0 100 0.47

Examples 47 and 48

[0082] Production of l-(1R,2R)-pseudoephedrine

[0083] Except that the microorganisms shown in Table 5 were used inplace of Amycolatopsis alba IFO 15602, optically active pseudoephedrinewas obtained similarly to Example 41. The produced amounts and opticalpurities of l-pseudoephedrine are shown in Table 5. TABLE 5 ProducedMicroorganism Optical purity (%) amount Example No. genus IFO1-pseudoephedrine (mg/ml) Example 47 Rhodococcus 12540 98.6 0.11erythropolis Example 48 Rhodococcus 15564 96.6 0.10 rhodochrous

Example 49

[0084] Production of l-(1R,2R)-pseudoephedrine

[0085]Coprinus rhizophorus IFO 30197 was inoculated to a mediumcontaining 1% glucose, 0.5% peptone and 0.3% yeast extract, andculturing was aerobically carried out at 30° C. for 48 hours. After thiscultured solution (5 ml) was centrifuged to give bacterial cells, thecells were dried in the air and the resulting dried bacterial cells weresuspended in 1 ml of 0.05 M Tris hydrochloric acid buffer (pH 7.5). Tothis were added glucose (50 mg), glucose dehydrogenase (0.2 mg), NADP(0.6 mg), NAD (0.6 mg) and dl-2-methylamino-1-phenyl-1-propanonehydrochloride (10 mg). Reaction was allowed to take place by carryingout reciprocation-shaking at 28° C. and at 300 rpm for 48 hours. Thereaction solution was analyzed on HPLC (Column Sumichiral AGPmanufactured by Sumika Chemical Analysis Service; diameter of 4 mm;length of 150 mm; 0.03 M sodium phosphate buffer; pH 7.0; flow rate of0.5 ml/min; detection wavelength at UV 220 nm). The produced amounts andthe optical activities of pseudoephedrine were determined. Consequently,only l-pseudoephedrine was obtained selectively, as shown in Table 6.TABLE 6 Optical purity (%) Produced Microorganism d- l- d-pseudo-l-pseudo- amount Example No. genus IFO ephedrine ephedrine ephedrineephedrine (mg/ml) Example 49 Corprinus 30197 0 0 0 100 1.09 rhizophorus

Example 50

[0086] Production of(1S,2S)-1-(p-hydroxyphenyl)-2-methylamino-1-propanol Rhodococcuserythropolis MAK-34 strain was shake-cultured in a medium (5 ml)containing 1% saccharose, 0.5% corn steep liquor, 0.1% potassiumdihydrogenphosphate, 0.3% dipotassium hydrogenphosphate and 0.1%1-amino-2-propanol at 30° C. for 48 hours. Either centrifugation orfiltration yielded bacterial cells. To this were added an adequateamount of water, 1M phosphate buffer (0.2 ml; pH 7.0), glucose (10 mg)and racemic 1-(p-hydroxyphenyl)-2-methylamino-1-propanone hydrochloride(1 mg), and they were mixed. One milliliter was shaken for reaction at30° C. for 48 hours. The reaction solution was either centrifuged orfiltered. The supernatant was analyzed on HPLC (μ Bondapakphenylmanufactured by Waters Inc.; diameter of 4 mm; length of 300 mm;eluent—0.05 M sodium phosphate buffer (containing 7% acetonitrile); pH6.5; flow rate of 0.8 ml/min; detection wavelength at UV 220 nm).Consequently, it was confirmed thatthreo-1-(p-hydroxyphenyl)-2-methylamino-1-propanol hydrochloride wasproduced at 0.6 mg/ml. To determine the optical purity of the product,the sample was analyzed on HPLC (Sumichiral OA-4900 manufactured bySumika Chemical Analysis Service; eluent:hexane:dichloroethane:methanol:trifluoroacetic acid=240:140:40:1; flow rate of 1 ml/min; detectionwavelength at UV 254 nm). Consequently, it was found that(1S,2S)-1-(p-hydroxyphenyl)-2-methylamino-1-propanol was obtained in100% optical purity.

Examples 51 to 54

[0087] Production of optically active1(p-hydroxyphenyl)-2-methylamino-1-propanol

[0088] Except that the microorganisms shown in Table 7 were used inplace of Rhodococcus erythropolis MAK-34 strain and the culturingconditions in the table were followed, reaction was carried outsimilarly to Example 50 and optically active1-(p-hydroxyphenyl)-2-methylamino-1-propanol was obtained. The resultsare shown in Table 7. In all instances, the optically active compoundwas obtained efficiently.

[0089] The culture conditions listed in Tables 7-9 are as follows:

[0090] Culture conditions 1: A microorganism was inoculated to a medium(20 ml) containing 1% glucose, 0.5% peptone, 10 and 0.3% yeast extract(pH7.0) and culturing was carried out at 30° C. for 48 hours under theshaking condition of 150 rpm.

[0091] Culture conditions 2: A microorganism was inoculated to a medium(20 ml, pH 6.0) containing 5% malt extract and 0.3% yeast extract andculturing was carried out at 30° C. for 48 hours under the shakingcondition of 150 rpm. TABLE 7 Produced Stereochemical Example Cultureamount configuration Optical No. Strain IFO conditions (mg/mL) ofproduct purity (%) Example 51 Helicostylum 8091 1 0.06 1S 2S 90nigricans Example 52 Amycolatopsis 14500 1 0.01 1R 2R 100 orientalislurida Example 53 Amycolatopsis 12362 1 0.02 1R 2R 100 orientalisorientalis Example 54 Amycolatopsis 12806 1 0.01 1R 2R 100 orientalisorientalis

Example 55

[0092] Production of (1S,2S)-2-ethylamino-1-phenyl-1-propanolRhodococcus erythropolis MAK-34 strain was shake-cultured in a medium (5ml) containing 1% saccharose, 0.5% corn steep liquor, 0.5% potassiumdihydrogenphosphate, 0.3% dipotassium hydrogenphosphate and 0.1%1-amino-2-propanol at 30° C. for 48 hours. Either centrifugation orfiltration yielded bacterial cells. To this were added an adequateamount of water, 1M phosphate buffer (0.2 ml, pH 7.0), glucose (10 mg)and racemic 2-ethylamino-1-phenyl-1-propanone hydrochloride (1 mg), andthey were mixed. One milliliter was shaken for reaction at 30° C. for 48hours. The reaction solution was either centrifuged or filtered. Thesupernatant was analyzed on HPLC (μ Bondaspherephenyl manufactured byWaters Inc.; diameter of 4 mm; length of 150 mm; eluent—0.05 M sodiumphosphate buffer (containing 7% acetonitrile); pH 6.5; flow rate of 0.8ml/min; detection wavelength at UV 220 nm). Consequently, it wasconfirmed that threo-2-ethylamino-1-phenyl-1-propanol hydrochloride wasproduced at 0.47 mg/ml. To determine the optical purity of the product,the sample was analyzed on HPLC (Column OD manufactured by DaicelChemical Industries Ltd.; diameter of 4.6 mm; length of 250 mm;eluent-hexane:isopropanol:diethylamine=90:10:0.1; flow rate of 1 ml/min;detection wavelength at UV 254 nm). Consequently, the product was foundto be (1S,2S)-2-ethylamino-1-phenyl-1-propanol (optical purity: 100%).

Examples 56 to 58

[0093] Production of (1R,2R)-2-ethylamino-1-phenyl-1-propanol

[0094] Except that the microorganisms shown in Table 8 were used inplace of Rhodococcus erythropolis MAK-34 strain and the culturingconditions in the table were followed, reaction was carried outsimilarly to Example 55 and (1R,2R)-2-ethylamino-1-phenyl-1-propanol wasobtained. The results are shown in Table 8. TABLE 8 ProducedStereochemical Example Culture amount configuration Optical No. StrainIFO conditions (mg/mL) of product purity (%) Example 56 Amycolatopsis15602 1 0.01 1R 2R 100 alba Example 57 Amycolatopsis 12806 1 0.01 1R 2R100 orientalis orientalis Example 58 Rhodotorula 0951 2 0.01 1R 2R 100aurantiaca

Examples 59 to 61

[0095] Production of Optically Active1-(m-hydroxyphenyl)-2-amino-1-propanol

[0096] The microorganisms listed in Table 9 were shake-cultured in amedium (5 ml) at 30° C. for 48 hours under their respective conditions.Either centrifugation or filtration yielded bacterial cells. To thiswere added an adequate amount of water, 1M phosphate buffer (0.2 ml, pH7.0), glucose (10 mg), and racemic1-(m-hydroxyphenyl)-2-amino-1-propanone hydrochloride (1 mg), and theywere mixed. One milliliter was shaken for reaction at 30° C. for 48hours. This was either centrifuged or filtered. The supernatant wasanalyzed on HPLC (μ Bondapakphenyl manufactured by Waters Inc.; diameterof 4 mm; length of 300 mm; eluent—0.05 M sodium phosphate buffer(containing 7% acetonitrile); pH 6.5; flow rate of 0.8 ml/min; detectionwavelength at UV 220 nm). Consequently, it was confirmed thatthreo-1-(m-hydroxyphenyl)-2-amino-1-propanol was produced. To determinethe optical purity of the product, the sample was analyzed on HPLC(Crownpak CR+ manufactured by Daicel Chemical Industries Ltd.;perchloric acid, pH 2.0, 1.0 ml/min, UV 254 nm). Consequently, it wasfound that optical active 1-(m-hydroxyphenyl)-2-amino-1-propanol withits stereochemical configuration shown in Table 9. TABLE 9 ProducedStereochemical Optical Example Culture amount configuration of purityNo. Strain IFO conditions (mg/mL) product (%) Example 59 Helicostylums8091 1 0.01 1S 2S 100 nigricans Example 60 Amycolatopsis 15602 1 0.01 1R2R 78 albas Example 61 Rhodotorula 0951 2 0.01 1R 2R 100 aurantiaca

Example 62

[0097] Production of (1R,2R)-1-(p-hydroxyphenyl)-2-amino-1-propanol

[0098]Amycolatopsis alba IFO-15602 was cultured under culture conditions1, and either centrifugation or filtration yielded bacterial cells. Tothese were added an adequate amount of water, 1M phosphate buffer (0.2ml, pH 7.0), glucose (10 mg) and racemic1-(p-hydroxyphenyl)-2-amino-1-propanone hydrochloride (1 mg), and theywere mixed. One milliliter was shaken for reaction at 30° C. for 48hours. This was either centrifuged or filtered. The supernatant wasanalyzed on HPLC (μ Bondapakphenyl manufactured by Waters Inc.; diameterof 4 mm; length of 300 mm; eluent-0.05 M sodium phosphate buffer(containing 7% acetonitrile); pH 6.5; flow rate of 0.8 ml/min; detectionwavelength at UV 220 nm). Consequently, it was confirmed thatthreo-1-(p-hydroxyphenyl)-2-amino-1-propanol hydrochloride was producedat 0.03 mg/ml. To determine the optical purity of the product, thesample was analyzed on HPLC (Crownpak CR+ manufactured by DaicelChemical Industries Ltd.; perchloric acid, pH 2.0, 1.0 ml/min, UV 254nm). Consequently, the product was found to be(1R,2R)-1-(p-hydroxyphenyl)-2-amino-1-propanol (optical purity: 82%).

Example 63

[0099] Production of (1S,2S)-1-phenyl-2-amino-1-butanol

[0100]Helicostylum nigricans IFO-8091 was cultured under cultureconditions 1. Either centrifugation or filtration yielded bacterialcells. To these were added an adequate amount of water, 1M phosphatebuffer (0.2 ml, pH 7.0), glucose (10 mg) and racemic1-phenyl-2-amino-1-butanone hydrochloride (1 mg), and they were mixed.One milliliter was shaken for reaction at 30° C. for 48 hours. Thereaction solution was either centrifuged or filtered. The supernatantwas analyzed on HPLC (μ Bondaspherephenyl manufactured by Waters Inc.;diameter of 4 mm; length of 150 mm; eluent—0.05 M sodium phosphatebuffer (containing 7% acetonitrile); pH 6.5; flow rate of 0.8 ml/min;detection wavelength at UV 220 nm). Consequently, it was found thatthreo-1-phenyl-2-amino-1-butanol hydrochloride was produced at 0.62mg/ml. To determine the optical purity of product, the sample wasanalyzed on HPLC (OD by Daicel Chemical Industries Ltd.; diameter of 4.6mm; length of 250 mm; hexane:isopropanol:diethylamine=90:10:0.1; 1ml/min; UV 254 nm). Consequently, the product was found to be(1S,2S)-1-phenyl-2-amino-1-butanol.

Example 64

[0101] Production of (1R,2R)-1-phenyl-2-amino-1butanol

[0102]Amycolatopsis orientalis IFO-12806 was cultured under cultureconditions 1, and similarly to Example 63,(1R,2R)-1-phenyl-2-amino-1-butanol could be produced at 0.21 mg/ml.

Example 65

[0103] The Effect of Addition of Inducer (1)

[0104] 1-Amino-2-hydroxypropanone was added to medium 1 (Table 10) sothat a level of 5 g/L could be obtained. Five milliliters was thenpoured into a test tube. With a silicone stopper it was sterilized in anautoclave at 121° C. for 30 minutes. The microorganisms listed in Table11 were inoculated to this medium and to the medium with no addition ofthe inducer, respectively; and they were shake-cultured at 300 rpm andat 30° C. for 48 hours. The culture (0.5 mL) was centrifuged at 10,000 Gfor 20 minutes. The bacterial cells obtained by removal of thesupernatant were suspended by addition of water to prepare a uniformsuspension. To this were added water, buffer anddl-2-methylamino-1-phenyl-1-propanone hydrochloride (10 mg), forming 1mL. The one milliliter was poured into a test tube and reaction wasallowed to take place under shaking at 150 rpm and at 30° C. for 12hours. After the reaction, the bacterial cells were removed bycentrifugation and the supernatant was subjected to HPLC, whereby theproduced amount of pseudoephedrine was determined (HPLC conditions: μBondapakphenyl manufactured by Waters Inc.; diameter of 4 mm; length of300 mm; eluent—0.05 M sodium phosphate buffer (containing 7%acetonitrile); pH 6.5; flow rate of 0.8 ml/min; detection wavelength atUV 220 nm).

[0105] As the results are shown in Table 11, the produced amounts ofpseudoephedrine when culturing was carried out with the addition of theinducer displayed remarkable increases as compared to the culturing withno addition of inducer. TABLE 10 Composition of Composition ofComposition of Composition of medium 1 medium 2 medium 3 medium 4saccharose 1% glucose 0.1% glucose 1% soluble starch 1% corn steeptryptone 0.5% Bactopeptone glucose 0.5% liquor 0.5% 0.5% potassium yeastyeast Nzaminetype dihydrogen- extract 0.5% extract 0.3% A 0.3% phosphate0.1% dipotassium dipotassium pH 7.0 tryptone 0.5% hydrogen- hydrogen-phosphate 0.3% phosphate p-aminobenzoic pH 7.0 yeast acid 0.01% extract0.2% H 7.0 dipotassium hydrogen- phosphate 0.1% magnesium sulfate 7H₂O0.05%

[0106] TABLE 11 Produced amount Produced (no amount addition) (addition)No. Microorganism No. Medium mg mg 1 Rhodococcus MAK-34 1 0.018 1.26erythropolis 2 Mycobacterium IFO-15527 3 0.032 0.77 chlorophenolicum 3Mycobacterium IFO-12065 3 0.048 0.21 smegmatis 4 Nocardioides IFO-120692 0 0.19 simplex 5 Klebsiella IFO-3319 2 0.018 0.066 pneumoniae 6Absidia IFO-4409 4 0.0035 0.22 lichtheimi 7 Aspergillus IFO-4033 40.00048 1.17 awamori 8 Aspergillus IFO-5468 4 0.0092 0.018 candidus 9Penicillium IFO-5337 4 0.031 1.26 cyaneum 10 Hypocrea IFO-9165 4 0.00580.64 gelatinosa 11 Helicostylum IFO-8091 4 0.0067 0.52 nigricans 12Tritirachium IFO-7544 4 0.0047 0.078 oryzae 13 Armillariella IFO-31616 40.0042 0.46 mellea

Example 66

[0107] The Effect of Addition of Inducers (2)

[0108]Rhodococcus erythropolis MAK-34 was inoculated to 5 ml of a medium(pH 7.0) containing 1.0% saccharose, 0.5% corn steep liquor, 0.1%dipotassium hydrogenphosphate, 0.3% potassium dihydrogenphosphate, 0.01%p-aminobenzoic acid and each inducer; and shake-culturing was carriedout at 30° C. for 48 hours. After the culture was centrifuged to givebacterial cells, they were placed into a test tube and suspended byadding 1.0 ml of 0.2 M sodium phosphate buffer (pH 7.0) thereto. To thiswere added dl-2-methylamino-1-phenyl-1-propanone hydrochloride (10 mg)and glucose (20 mg) and reaction was allowed to take place under shakingat 30° C. for 16 hours. After the reaction, the reaction solution wascentrifuged to remove bacterial cells, and the supernatant was subjectedto HPLC, producing optically active pseudoephedrine (μ Bondaspherephenylmanufactured by Waters Inc.; diameter of 4 mm; length of 150 mm;eluent—7% acetonitrile-0.05 M sodium phosphate buffer (pH 6.5); flowrate of 0.8 ml/min; detection wavelength at 220 nm). As shown in Table12, the production displayed remarkably higher values than does the casewithout the addition of inducer. TABLE 12 Compound name Produced amount(mg) 1-acetylamino-2-propanol 3.00 1-methylamino-2-propanol 2.831-amino-2-oxopropane 1.97 2-amino-3-hydroxybutane 0.051-amino-2-hydroxybutane 0.65 no addition 0.02

Comparative Example 1

[0109] Except that Brettanomyces anomalus IFO 0642 was used instead ofMicrobacterium arborescens IFO 3750, the production reaction ofpseudoephedrine was attempted similarly to Example 1. However, noreduced product was obtained.

Comparative Example 2

[0110] Except that Candida guilliermondii IFO 0566 was used instead ofMicrobacterium arborescens IFO 3750, the production reaction ofpseudoephedrine was attempted similarly to Example 1. However, noreduced product was obtained.

Comparative Example 3

[0111] Except that Schizosaccharomyces pombe IFO 0358 was used insteadof Microbacterium arborescens IFO 3750, the production reaction ofpseudoephedrine was attempted similarly to Example 1. However, noreduced product was obtained.

Comparative Example 4

[0112] Except that Bacillus subtilis IFO 3037 was used instead ofMicrobacterium arborescens IFO 3750, the production reaction ofpseudoephedrine was attempted similarly to Example 1. However, noreduced product was obtained.

Industrial Applicability

[0113] As described above, the process for producing an optically activeβ-amino alcohol according to this invention allows the β-amino alcoholhaving the desired optical activity to be produced from an enantiomericmixture of an α-aminoketone compound or a salt thereof in a high yieldas well as in a highly selective manner with a simple process whilesufficiently preventing the generation of diastereomeric byproducts.

[0114] Accordingly, this invention will make it possible to producepseudoephedrines, among others, having the desired optical activity in ahigh yield as well as in a highly selective manner and thus it isvaluable in the manufacture of drugs and their intermediates.

1. A process for producing an optical active β-amino alcohol, theprocess comprising allowing at least one microorganism selected from thegroup consisting of microorganisms belonging to the genus Morganellagenus, the genus Microbacterium, the genus Sphingobacterium, the genusNocardioides, the genus Mucor, the genus Absidia, the genus Aspergillus,the genus Penicillium, the genus Grifola, the genus Eurotium, the genusGanoderma, the genus Hypocrea, the genus Helicostylum, the genusVerticillium, the genus Fusarium, the genus Tritirachium, the genusMortierella, the genus Armillariella, the genus Cylindrocarpon, thegenus Klebsiella, the genus Aureobacterium, the genus Xanthomonas, thegenus Pseudomonas, the genus Mycobacterium, the genus Sporobolomyces,the genus Sporidiobolus, the genus Amycolatopsis, the genus Coprinus,the genus Serratia, the genus Rhodococcus and the genus Rhodotorula toact on an enantiomeric mixture of an α-amino ketone or a salt thereofhaving the general formula (I):

wherein X may be the same or different and represents at least onemember selected from the group consisting of a halogen atom, loweralkyl, hydroxyl optionally protected with a protecting group, nitro andsulfonyl; n represents an integer of from 0 to 3; R¹ represents loweralkyl; R² and R³ may be the same or different and represent at least onemember selected from the group consisting of a hydrogen atom and loweralkyl; and “*” represents an asymmetric carbon, to produce an opticallyactive β-amino alcohol compound with the desired optical activity havingthe general formula (II):

wherein X, n, R¹, R², R³ and “*” are as previously defined.
 2. Theprocess for producing an optically active β-amino alcohol according toclaim
 1. wherein the microorganism is at least one microorganismselected from the group consisting of microorganisms belonging toMorganella morganii, Microbacterium arborescens, Sphingobacteriummultivorum, Nocardioides simplex, Mucor ambiguus, Mucor javanicus, Mucorfragilis, Absidia lichtheimi, Aspergillus awamori, Aspergillus niger,Aspergillus oryzae, Aspergillus candidus, Aspergillus oryzae var.oryzae, Aspergillus foetidus var. acidus, Penicillium oxalicum, Grifolafrondosa, Eurotium repens, Ganoderma lucidum, Hypocrea gelatinosa,Helicostylum nigricans, Verticillium fungicola var. fungicola, Fusariumroseum, Tritirachium oryzae, Mortierella isabellina, Armillariellamellea, Cylindrocarpon sclerotigenum, Klebsiella pneumoniae,Aureobacterium esteraromaticum, Xanthomonas sp., Pseudomonas putida,Mycobacterium smegmatis, Mycobacterium diernhoferi, Mycobacteriumvaccae, Mycobacterium phlei, Mycobacterium fortuitum, Mycobacteriumchlorophenolicum, Sporobolomyces salmonicolor, Sporobolomycescoralliformis, Sporidiobolus johnsonii, Amycolatopsis alba,Amycolatopsis azurea, Amycolatopsis coloradensis, Amycolatopsisorientalis lurida, Amycolatopsis orientalis orientalis, Coprinusrhizophorus, Serratia marcescens, Rhodococcus erythropolis, Rhodococcusrhodochrous and Rhodotorula aurantiaca.
 3. The process for producing anoptically active β-amino alcohol according to claim 1, wherein themicroorganism is at least one microorganism selected from the groupconsisting of microorganisms belonging to the genus Morganella, thegenus Microbacterium, the genus Sphingobacterium, the genusNocardioides, the genus Mucor, the genus Absidia, the genus Aspergillus,the genus Penicillium, the genus Grifola, the genus Eurotium, the genusGanoderma, the genus Hypocrea, the genus Helicostylum, the genusVerticillium, the genus Fusarium, the genus Tritirachium, the genusMortierella, the genus Armillariella, the genus Cylindrocarpon, thegenus Klebsiella, the genus Aureobacterium, the genus Xanthomonas, thegenus Pseudomonas, the genus Mycobacterium, the genus Sporobolomyces,the genus Sporidiobolus and the Rhodococcus genus; and the β-aminoalcohol having the general formula (II) is (1S,2S)-amino alcohol.
 4. Theprocess for producing an optically active β-amino alcohol according toclaim 3, wherein the microorganism is at least one microorganismselected from the group consisting of microorganisms belonging toMorganella morganii, Microbacterium arborescens, Sphingobacteriummultivorum, Nocardioides simplex, Mucor ambiguus, Mucor javanicus, Mucorfragilis, Absidia lichtheimi, Aspergillus awamori, Aspergillus niger,Aspergillus oryzae, Aspergillus candidus, Aspergillus oryzae var.oryzae, Aspergillus foetidus var. acidus, Penicillium oxalicum, Grifolafrondosa, Eurotium repens, Ganoderma lucidum, Hypocrea gelatinosa,Helicostylum nigricans, Verticillium fungicola var. fungicola, Fusariumroseum, Tritirachium oryzae, Mortierella isabellina, Armillariellamellea, Cylindrocarpon sclerotigenum, Klebsiella pneumoniae,Aureobacterium esteraromaticum, Xanthomonas sp., Pseudomonas putida,Mycobacterium smegmatis, Mycobacterium diernhoferi, Mycobacteriumvaccae, Mycobacterium phlei, Mycobacterium fortuitum, Mycobacteriumchlorophenolicum, Sporobolomyces salmonicolor, Sporobolomycescoralliformis, Sporidiobolus johnsonii, Rhodococus erythropolis andRhodococcus rhodochrous.
 5. The process for producing an opticallyactive β-amino alcohol according to claim 1, wherein the microorganismis at least one microorganism selected from the group consisting ofmicroorganisms belonging to the genus Amycolaptopsis, the genusCoprinus, the genus Serratia, the genus Rhodococcus and the genusRhodotorula; and the optically active β-amino alcohol having the generalformula (II) is (1R,2R)-amino alcohol.
 6. The process for producing anoptically active β-amino alcohol according to claim 5, wherein themicroorganism is at least one microorganism selected from the groupconsisting of microorganisms belonging to Amycolatopsis alba,Amycolatopsis azurea, Amycolatopsis coloradensis, Amycolatopsisorientalis lurida, Amycolatopsis orientalis orientalis, Coprinusrhizophorus, Serratia marcescens, Rhodococcus erythropolis, Rhodococcusrhodochrous and Rhodotorula aurantiaca.
 7. The process for producing anoptically active β-amino alcohol according to any of claims 1-6, whereinthe microorganism is cultured in a medium to which there has been addedan activity inducer having the general formula (III):

wherein R⁴ represents lower alkyl; R⁵ and R⁶ may be the same ordifferent and each represents a hydrogen atom, lower alkyl or acyl; andY represents C═O or CH—OH.